Depletion of E-cadherin disrupts establishment but not maintenance of cell junctions in Madin-Darby canine kidney epithelial cells

E-cadherin forms calcium-dependent homophilic intercellular adhesions between epithelial cells. These contacts regulate multiple aspects of cell behavior, including the organization of intercellular tight junctions (TJs). To distinguish between the roles of E-cadherin in formation versus maintenance of junctions, Madin-Darby canine kidney (MDCK) cells were depleted of E-cadherin by RNA interference. Surprisingly, reducing E-cadherin expression had little effect on the protein levels or localization of adherens junction (AJ) or TJ markers. The cells underwent morphological changes, as the normally flat apical surface swelled into a dome. However, apical-basal polarity was not compromised, transmembrane resistance was normal, and zonula occludin protein 1 dynamics at the TJs were unchanged. Additionally, an E-cadherin/Cadherin-6 double knockdown also failed to disrupt established TJs, although beta-catenin was lost from the cell cortex. Nevertheless, cells depleted of E-cadherin failed to properly reestablish cell polarity after junction disassembly. Recovery of cell-cell adhesion, transepithelial resistance, and the localization of TJ and AJ markers were all delayed. In contrast, depletion of alpha-catenin caused long-term disruption of junctions. These results indicate that E-cadherin and Cadherin-6 function as a scaffold for the construction of polarized structures, and they become largely dispensable in mature junctions, whereas alpha-catenin is essential for the maintenance of functional junctions.     

E-Cadherin Is Required for Centrosome and Spindle Orientation in Drosophila Male Germline Stem Cells

Many adult stem cells reside in a special microenvironment known as the niche, where they receive essential signals that specify stem cell identity. Cell-cell adhesion mediated by cadherin and integrin plays a crucial role in maintaining stem cells within the niche. In Drosophila melanogaster, male germline stem cells (GSCs) are attached to niche component cells (i.e., the hub) via adherens junctions. The GSC centrosomes and spindle are oriented toward the hub-GSC junction, where E-cadherin-based adherens junctions are highly concentrated. For this reason, adherens junctions are thought to provide a polarity cue for GSCs to enable proper orientation of centrosomes and spindles, a critical step toward asymmetric stem cell division. However, understanding the role of E-cadherin in GSC polarity has been challenging, since GSCs carrying E-cadherin mutations are not maintained in the niche. Here, we tested whether E-cadherin is required for GSC polarity by expressing a dominant-negative form of E-cadherin. We found that E-cadherin is indeed required for polarizing GSCs toward the hub cells, an effect that may be mediated by Apc2. We also demonstrated that E-cadherin is required for the GSC centrosome orientation checkpoint, which prevents mitosis when centrosomes are not correctly oriented. We propose that E-cadherin orchestrates multiple aspects of stem cell behavior, including polarization of stem cells toward the stem cell-niche interface and adhesion of stem cells to the niche supporting cells.     

Mechanosensitive Adaptation of E-Cadherin Turnover across adherens Junctions

In the natural and technological world, multi-agent systems strongly depend on how the interactions are ruled between their individual components, and the proper control of time-scales and synchronization is a key issue. This certainly applies to living tissues when multicellular assemblies such as epithelial cells achieve complex morphogenetic processes. In epithelia, because cells are known to individually generate actomyosin contractile stress, each individual intercellular adhesive junction line is subjected to the opposed stresses independently generated by its two partner cells. Contact lines should thus move unless their two partner cells mechanically match. The geometric homeostasis of mature epithelia observed at short enough time-scale thus raises the problem to understand how cells, if considered as noisy individual actuators, do adapt across individual intercellular contacts to locally balance their time-average contractile stress. Structural components of adherens junctions, cytoskeleton (F-actin) and homophilic bonds (E-cadherin) are quickly renewed at steady-state. These turnovers, if they depend on forces exerted at contacts, may play a key role in the mechanical adaptation of epithelia. Here we focus on E-cadherin as a force transducer, and we study the local regulation and the mechanosensitivity of its turnover in junctions. We show that E-cadherin turnover rates match remarkably well on either side of mature intercellular contacts, despite the fact that they exhibit large fluctuations in time and variations from one junction to another. Using local mechanical and biochemical perturbations, we find faster turnover rates with increased tension, and asymmetric rates at unbalanced junctions. Together, the observations that E-cadherin turnover, and its local symmetry or asymmetry at each side of the junction, are mechanosensitive, support the hypothesis that E-cadherin turnover could be involved in mechanical homeostasis of epithelia.     

Regulation of adherens junctions and epithelial paracellular permeability: a novel function for polyamines

Maintenance of intestinal mucosal epithelial integrity requires polyamines that are involved in the multiple signaling pathways controlling gene expression and different epithelial cell functions. Integrity of the intestinal epithelial barrier depends on a complex of proteins composing different intercellular junctions, including tight junctions, adherens junctions, and desmosomes. E-cadherin is primarily found at the adherens junctions and plays a critical role in cell-cell adhesions that are fundamental to formation of the intestinal epithelial barrier. The current study determined whether polyamines regulate intestinal epithelial barrier function by altering E-cadherin expression. Depletion of cellular polyamines by alpha-difluoromethylornithine (DFMO) reduced intracellular free Ca2+ concentration ([Ca2+]cyt), decreased E-cadherin expression, and increased paracellular permeability in normal intestinal epithelial cells (IEC-6 line). Polyamine depletion did not alter expression of tight junction proteins such as zona occludens (ZO)-1, ZO-2, and junctional adhesion molecule (JAM)-1. Addition of exogenous polyamine spermidine reversed the effects of DFMO on [Ca2+]cyt and E-cadherin expression and restored paracellular permeability to near normal. Elevation of [Ca2+]cyt by the Ca2+ ionophore ionomycin increased E-cadherin expression in polyamine-deficient cells. In contrast, reduction of [Ca2+]cyt by polyamine depletion or removal of extracellular Ca2+ not only inhibited expression of E-cadherin mRNA but also decreased the half-life of E-cadherin protein. These results indicate that polyamines regulate intestinal epithelial paracellular barrier function by altering E-cadherin expression and that polyamines are essential for E-cadherin expression at least partially through [Ca2+]cyt.     

Caveolae Mediate Growth Factor-induced Disassembly of Adherens Junctions to Support Tumor Cell Dissociation

Remodeling of cell–cell contacts through the internalization of adherens junction proteins is an important event during both normal development and the process of tumor cell metastasis. Here we show that the integrity of tumor cell–cell contacts is disrupted after epidermal growth factor (EGF) stimulation through caveolae-mediated endocytosis of the adherens junction protein E-cadherin. Caveolin-1 and E-cadherin closely associated at cell borders and in internalized structures upon stimulation with EGF. Furthermore, preventing caveolae assembly through reduction of caveolin-1 protein or expression of a caveolin-1 tyrosine phospho-mutant resulted in the accumulation of E-cadherin at cell borders and the formation of tightly adherent cells. Most striking was the fact that exogenous expression of caveolin-1 in tumor cells that contain tight, well-defined, borders resulted in a dramatic dispersal of these cells. Together, these findings provide new insights into how cells might disassemble cell–cell contacts to help mediate the remodeling of adherens junctions, and tumor cell metastasis and invasion.     

Zonula occludens-1 and E-cadherin are coordinately expressed in the mouse uterus with the initiation of implantation and decidualization

Two-way interactions between the blastocyst trophectoderm and the uterine luminal epithelium are essential for implantation. The key events of this process are cell-cell contact of trophectoderm cells with uterine luminal epithelial cells, controlled invasion of trophoblast cells through the luminal epithelium and the basement membrane, transformation of uterine stromal cells surrounding the blastocyst into decidual cells, and protection of the "semiallogenic" embryo from the mother's immunological responses. Because cell-cell contact between the trophectoderm epithelium and the luminal epithelium is essential for implantation, we investigated the expression of zonula occludens-1 (ZO-1) and E-cadherin, two molecules associated with epithelial cell junctions, in the mouse uterus during the periimplantation period. Preimplantation uterine epithelial cells express both ZO-1 and E-cadherin. With the initiation and progression of implantation, ZO-1 and E-cadherin are expressed in stromal cells of the primary decidual zone (PDZ). As trophoblast invasion progresses, these two molecules are expressed in stroma in advance of the invading trophoblast cells. These results suggest that expression of these adherence and tight junctions molecules in the PDZ serves to function as a permeability barrier to regulate access of immunologically competent maternal cells and/or molecules to the embryo and provide homotypic guidance of trophoblast cells in the endometrium.     

An association between type Iγ PI4P 5-kinase and Exo70 directs E-cadherin clustering and epithelial polarization

E-Cadherin-mediated formation of adherens junctions (AJs) is essential for the morphogenesis of epithelial cells. However, the mechanisms underlying E-cadherin clustering and AJ maturation are not fully understood. Here we report that type Iγ phosphatidylinositol-4-phosphate 5-kinase (PIPKIγ) associates with the exocyst via a direct interaction with Exo70, the exocyst subunit that guides the polarized targeting of exocyst to the plasma membrane. By means of this interaction, PIPKIγ mediates the association between E-cadherin and Exo70 and determines the targeting of Exo70 to AJs. Further investigation revealed that Exo70 is necessary for clustering of E-cadherin on the plasma membrane and extension of nascent E-cadherin adhesions, which are critical for the maturation of cohesive AJs. In addition, we observed phosphatidylinositol-4,5-bisphosphate (PI4,5P(2)) accumulation at E-cadherin clusters during the assembly of E-cadherin adhesions. PIPKIγ-generated PI4,5P(2) is required for recruiting Exo70 to newly formed E-cadherin junctions and facilitates the assembly and maturation of AJs. These results support a model in which PIPKIγ and PIPKIγ-generated PI4,5P(2) pools at nascent E-cadherin contacts cue Exo70 targeting and orient the tethering of exocyst-associated E-cadherin. This could be an important mechanism that regulates E-cadherin clustering and AJ maturation, which is essential for the establishment of solid, polarized epithelial structures.     

Regulation of Cadherin Function by Rho and Rac: Modulation by Junction Maturation and Cellular Context

Cadherins are cell–cell adhesion receptors whose adhesive function requires their association with the actin cytoskeleton via proteins called catenins. The small guanosine triphosphatases (GTPases), Rho and Rac, are intracellular proteins that regulate the formation of distinct actin structures in different cell types. In keratinocytes and in other epithelial cells, Rho and Rac activities are required for E-cadherin function. Here we show that the regulation of cadherin adhesiveness by the small GTPases is influenced by the maturation status of the junction and the cellular context. E-cadherin localization was disrupted in mature keratinocyte junctions after inhibition of Rho and Rac. However, an incubation of 2 h was required after GTPase inhibition, when compared with newly established E-cadherin contacts (30 min). Regarding other cadherin receptors, P-cadherin was effectively removed from mature keratinocytes junctions by blocking Rho or Rac. In contrast, VE-cadherin localization at endothelial junctions was independent of Rho/Rac activity. We demontrate that the insensitivity of VE-cadherin to inhibition of Rho and Rac was not due to the maturation status of endothelial junction, but rather the cellular background: when transfected into CHO cells, the localization of VE-cadherin was perturbed by inhibition of Rho proteins. Our results suggest that the same stimuli may have different activity in regulating the paracellular activity in endothelial and epithelial cells. In addition, we uncovered possible roles for the small GTPases during the establishment of E-cadherin–dependent contacts. In keratinocytes, Rac activation by itself cannot promote accumulation of actin at the cell periphery in the absence of cadherin-dependent contacts. Moreover, neither Rho nor Rac activation was sufficient to redistribute cadherin molecules to cell borders, indicating that redistribution results mostly from the homophilic binding of the receptors. Our results point out the complexity of the regulation of cadherin-mediated adhesion by the small GTPases, Rho and Rac.     

Cell junctions in early embryos of squid (Loligo pealei)

Summary Squid embryos examined by freeze-fracture and thin-section electron microscopy exhibit identifiable gap junctions during mid-cleavage stages (stages 7–8), and junctional complexes composed of adherent appositions, elaborate septate junctions and gap junctions at slightly later stages (stages 12–13). During germinal layer establishment (stages 12–13) cytoplasmic bridges frequently link the embryonic cells. The presence of gap junctions in cleavagestage embryos provides the morphological substrate for a demonstrated pathway of direct cell-cell communication that is modifiable by experimental treatments and may be physiologically regulatable. The existence of septate junctions and adherent contacts at later stages suggests that some functional specialization, perhaps the establishment of a strongly joined framework of cells at the surface of the embryo, accompanies the formation of germinal layers.     

Tumor suppressor gene E-cadherin and its role in normal and malignant cells

E-cadherin tumor suppressor genes are particularly active area of research in development and tumorigenesis. The calcium-dependent interactions among E-cadherin molecules are critical for the formation and maintenance of adherent junctions in areas of epithelial cell-cell contact. Loss of E-cadherin-mediated-adhesion characterises the transition from benign lesions to invasive, metastatic cancer. Nevertheless, there is evidence that E-cadherins may also play a role in the wnt signal transduction pathway, together with other key molecules involved in it, such as beta-catenins and adenomatous poliposis coli gene products.     

Cytoarchitectonics and intercellular relations during posttraumatic regeneration of the epidermis

Dynamics of changes of intercellular contacts in the mouse spine epidermis have been studied in 1, 3, 5, 7, 10 days after its traumatic lesion performed up to the upper level of the papillary layer of the derm. In one day a complete desintegration of basal keratinocytes with some signs of reactive changes takes place. A partial desintegration is noted in the exfoliated spinous layer, which participates in the crust formation, as well as in formation of cells in marginal zones of the defect. When there is an intercellular edema, desmosomes manifest themselves as the most highly-adhesive junctions. On the 1st-7th day melanocytes loose their contacts with keratinocytes, but do not display any reactive changes. On the 7th day hyperplasia of the epithelial layer is noted with an increased number of apigmented granular dendrocytes that participate in fagocytosis. The intercellular contacts are reconstructed, however, essential intercellular spaces remain. By the 10th day the contacting surfaces of the cells are completely formed, possessing all specific for epidermis specialized junctions. Columnar organization of the layer is formed.     

E-cadherin dis-engagement activates the Rap1 GTPase

E-cadherin based adherens junctions are finely regulated by multiple cellular signaling events. Here we show that the Ras-related Rap1 GTPase is enriched in regions of nascent cell-cell contacts and strengthens E-cadherin junctions: constitutively active Rap1 expressing MDCK cells exhibit increased junctional contact and resisted calcium depletion-induced cell-cell junction disruption. E-cadherin disengagement activated Rap1 and this correlated with E-cadherin association with the Rap GEFs, C3G and PDZ-GEF I. PDZ-GEF I associated with E-cadherin and beta-catenin whereas C3G interaction with E-cadherin did not involve beta-catenin. Knockdown of PDZ-GEF I in MDCK cells decreased Rap1 activity following E-cadherin junction disruption. We hereby show that Rap1 plays a role in the maintenance and repair of E-cadherin junctions and is activated via an "outside-in" signaling pathway initiated by E-cadherin and mediated at least in part by PDZ-GEF I.     

Presenilin-1 forms complexes with the cadherin/catenin cell-cell adhesion system and is recruited to intercellular and synaptic contacts

In MDCK cells, presenilin-1 (PS1) accumulates at intercellular contacts where it colocalizes with components of the cadherin-based adherens junctions. PS1 fragments form complexes with E-cadherin, beta-catenin, and alpha-catenin, all components of adherens junctions. In confluent MDCK cells, PS1 forms complexes with cell surface E-cadherin; disruption of Ca(2+)-dependent cell-cell contacts reduces surface PS1 and the levels of PS1-E-cadherin complexes. PS1 overexpression in human kidney cells enhances cell-cell adhesion. Together, these data show that PS1 incorporates into the cadherin/catenin adhesion system and regulates cell-cell adhesion. PS1 concentrates at intercellular contacts in epithelial tissue; in brain, it forms complexes with both E- and N-cadherin and concentrates at synaptic adhesions. That PS1 is a constituent of the cadherin/catenin complex makes that complex a potential target for PS1 FAD mutations.     

E-cadherin is essential for in vivo epidermal barrier function by regulating tight junctions

Cadherin adhesion molecules are key determinants of morphogenesis and tissue architecture. Nevertheless, the molecular mechanisms responsible for the morphogenetic contributions of cadherins remain poorly understood in vivo. Besides supporting cell-cell adhesion, cadherins can affect a wide range of cellular functions that include activation of cell signalling pathways, regulation of the cytoskeleton and control of cell polarity. To determine the role of E-cadherin in stratified epithelium of the epidermis, we have conditionally inactivated its gene in mice. Here we show that loss of E-cadherin in the epidermis in vivo results in perinatal death of mice due to the inability to retain a functional epidermal water barrier. Absence of E-cadherin leads to improper localization of key tight junctional proteins, resulting in permeable tight junctions and thus altered epidermal resistance. In addition, both Rac and activated atypical PKC, crucial for tight junction formation, are mislocalized. Surprisingly, our results indicate that E-cadherin is specifically required for tight junction, but not desmosome, formation and this appears to involve signalling rather than cell contact formation.     

Induction of polarized cell-cell association and retardation of growth by activation of the E-cadherin-catenin adhesion system in a dispersed carcinoma line

PC9 lung carcinoma cells cannot tightly associate with one another, and therefore grow singly, despite their expression of E-cadherin, because of their lack of alpha-catenin, a cadherin-associated protein. However, when the E-cadherin is activated by transfection with alpha-catenin cDNA, they form spherical aggregates, each consisting of an enclosed monolayer cell sheet. In the present work, we examined whether the alpha-catenin-transfected cell layers expressed epithelial phenotypes, by determining the distribution of various cell adhesion molecules on their surfaces, including E-cadherin, ZO-1, desmoplakin, integrins, and laminin. In untransfected PC9 cells, all these molecules were randomly distributed on their cell surface. In the transfected cells, however, each of them was redistributed into a characteristic polarized pattern without a change in the amount of expression. Electron microscopic study demonstrated that the alpha-catenin-transfected cell layers acquired apical-basal polarity typical of simple epithelia; they formed microvilli only on the outer surface of the aggregates, and a junctional complex composed of tight junction adherens junction, and desmosome arranged in this order. These results indicate that the activation of E-cadherin triggered the formation of the junctional complex and the polarized distribution of cell surface proteins and structures. We also found that, in untransfected PC9 cells, ZO-1 formed condensed clusters and colocalized with E-cadherin, but that other adhesion molecules rarely showed such colocalization with E-cadherin, suggesting that there is some specific interaction between ZO-1 and E- cadherin even in the absence of cell-cell contacts. In addition, we found that the activation of E-cadherin caused a retardation of PC9 cell growth. Thus, we concluded that the E-cadherin-catenin adhesion system is essential not only for structural organization of epithelial cells but also for the control of their growth.     

Dynamics of adherens junctions in epithelial establishment, maintenance, and remodeling

The epithelial cadherin (E-cadherin)-catenin complex binds to cytoskeletal components and regulatory and signaling molecules to form a mature adherens junction (AJ). This dynamic structure physically connects neighboring epithelial cells, couples intercellular adhesive contacts to the cytoskeleton, and helps define each cell's apical-basal axis. Together these activities coordinate the form, polarity, and function of all cells in an epithelium. Several molecules regulate AJ formation and integrity, including Rho family GTPases and Par polarity proteins. However, only recently, with the development of live-cell imaging, has the extent to which E-cadherin is actively turned over at junctions begun to be appreciated. This turnover contributes to junction formation and to the maintenance of epithelial integrity during tissue homeostasis and remodeling.     

Gap junction protein connexin-43 interacts directly with microtubules.

Gap junctions are specialized cell-cell junctions that mediate intercellular communication. They are composed of connexin proteins, which form transmembrane channels for small molecules [1, 2]. The C-terminal tail of connexin-43 (Cx43), the most widely expressed connexin member, has been implicated in the regulation of Cx43 channel gating by growth factors [3-5]. The Cx43 tail contains various protein interaction sites, but little is known about binding partners. To identify Cx43-interacting proteins, we performed pull-down experiments using the C-terminal tail of Cx43 fused to glutathione-S-transferase. We find that the Cx43 tail binds directly to tubulin and, like full-length Cx43, sediments with microtubules. Tubulin binding to Cx43 is specific in that it is not observed with three other connexins. We established that a 35-amino acid juxtamembrane region in the Cx43 tail, which contains a presumptive tubulin binding motif, is necessary and sufficient for microtubule binding. Immunofluorescence and immunoelectron microscopy studies reveal that microtubules extend to Cx43-based gap junctions in contacted cells. However, intact microtubules are dispensable for the regulation of Cx43 gap-junctional communication. Our findings suggest that, in addition to its well-established role as a channel-forming protein, Cx43 can anchor microtubule distal ends to gap junctions and thereby might influence the properties of microtubules in contacted cells.     

FRAP Analysis Reveals Stabilization of Adhesion Structures in the Epidermis Compared to Cultured Keratinocytes

Proper development and tissue maintenance requires cell-cell adhesion structures, which serve diverse and crucial roles in tissue morphogenesis. Epithelial tissues have three main types of cell-cell junctions: tight junctions, which play a major role in barrier formation, and adherens junctions and desmosomes, which provide mechanical stability and organize the underlying cytoskeleton. Our current understanding of adhesion function is hindered by a lack of tools and methods to image junctions in mammals. To better understand the dynamics of adhesion in tissues we have created a knock-in ZO-1-GFP mouse and a BAC-transgenic mouse expressing desmoplakin I-GFP. We performed fluorescence recovery after photobleaching (FRAP) experiments to quantify the turnover rates of the tight junction protein ZO-1, the adherens junction protein E-cadherin, and the desmosomal protein desmoplakin in the epidermis. Proteins at each type of junction are remarkably stable in the epidermis, in contrast to the high observed mobility of E-cadherin and ZO-1 at adherens junctions and tight junctions, respectively, in cultured cells. Our data demonstrate that there are additional mechanisms for stabilizing junctions in tissues that are not modeled by cell culture.     

Src-induced de-regulation of E-cadherin in colon cancer cells requires integrin signalling

Although Src expression and activity are often elevated in colon cancer, the precise consequences of overexpression of the non-catalytic Src homology (SH) domains, or enhanced catalytic activity, are unknown. We show that, in KM12C colon cancer cells, elevated Src activity causes the components of adherens junctions, including vinculin, to be redistributed to Src-induced integrin adhesion complexes. Specifically, elevated Src activity blocks proper assembly of cell cell contacts after cells are switched from media containing a low level of calcium to media containing a high level of calcium, and E-cadherin remains internalized. In contrast, although elevated expression of the non-catalytic domains of Src is sufficient to induce assembly of integrin adhesion complexes, it does not induce disorganization of E-cadherin-associated intercellular contacts. Surprisingly, Src-induced disruption of E-cadherin localization requires specific integrin signalling, because E-cadherin redistribution is blocked by loss of cell-matrix interaction, or by inhibitory antibodies to alpha(v) or beta(1) integrin subunits. Furthermore, phosphorylation of the integrin-regulated focal adhesion kinase (FAK) on Src-specific sites is required for Src-induced de-regulation of E-cadherin, demonstrating interdependence between integrin-induced signals and cadherin-associated adhesion changes induced by Src.